Differential inflammasome expression and IL-1β secretion in monocyte-derived dendritic cells differentiated with IL-4 or IFN-α

NLRP3-inflammasome activation was evaluated in monocyte-derived dendritic cells (DC) obtained through IL-4 (IL4-DC) or IFN-α (IFN-DC) protocols and pulsed with chemically inactivated HIV-1. Inflammasome' genes expression and IL-1β secretion were compared in DC isolated from 15 healthy subjects (HC) and 10 HIV-1 infected individuals (HIV+).\ud \ud FINDINGS:\ud Whether HIV was able to increased NLRP3-inflammasome genes expression and IL-1β secretion in IL4-DC from HC, the induction of inflammasome appeared significantly reduced in IFN-DC from HC, suggesting a different responsive state of IFN-DC compared to IL4-DC. No inflammasome activation was observed in IL4-DC as well as in IFN-DC derived from HIV + subjects, confirming previous findings on "unresponsive" state of DC derived from HIV + possibly due to chronic inflammatory state of these individuals.\ud \ud CONCLUSIONS:\ud Our results showed that IFN-α differently modulates inflammasome expression during monocytes-DC in vitro differentiation. These findings could be of interest considering the on-going research about DC manipulation and therapeutic strategies for HIV + involving DC-based immune-vaccines.Sao Paulo Research Foundation (FAPESP

Using Dendritic Cell-Based Immunotherapy to Treat HIV: How Can This Strategy be Improved?

Harnessing dendritic cells (DC) to treat HIV infection is considered a key strategy to improve anti-HIV treatment and promote the discovery of functional or sterilizing cures. Although this strategy represents a promising approach, the results of currently published trials suggest that opportunities to optimize its performance still exist. In addition to the genetic and clinical characteristics of patients, the efficacy of DC-based immunotherapy depends on the quality of the vaccine product, which is composed of precursor-derived DC and an antigen for pulsing. Here, we focus on some factors that can interfere with vaccine production and should thus be considered to improve DC-based immunotherapy for HIV infection

Immunophenotypic and functional characterization of IFN-DC derived HIV-1 infected patients.

A imunoterapia baseada em MoDC constitui uma estratégia para tratamento de indivíduos HIV+. Protocolos para obtenção de MoDC em geral utilizam IL-4 e GM-CSF (IL4-DC). Alguns estudos utilizam as IFN-DC (IFN-α + GM-CSF), que exibem um fenótipo combinado de DC mielóide, DC plasmocitóide (pDC) e célula NK. Esse perfil misto pode aperfeiçoar a imunoterapia para pacientes HIV+. Para tanto, monócitos de pacientes HIV+ foram cultivados com GM-CSF e IL-4 ou IFN-α por 5 dias e estimuladas por 48 horas com pulso de HIV inativado por AT-2 e/ou coquetel de citocinas pró-inflamatórias. Avaliamos a expressão de moléculas de superfície de IFN-DC e ativação de linfócitos T por citometria de fluxo; produção de citocinas IL-12 e IL-10 por ELISA. IFN-DC apresentaram morfologia e fenótipo basais ativados e características de pDC e célula NK, diferente das IL4-DC. As IFN-DC foram capazes de produzir IL-12, estimular a proliferação e produção de IFN-γ de linfócitos TCD4 e CD8, porém similares às IL4-DCs. IFN-DC são capazes de estimular resposta de linfócitos T tanto quanto IL4-DC.Immunotherapy based on MDDCs is a strategy for treating HIV-infected patients. Alternatively to the conventional protocol for DC differentiation based on IL-4 and GM-CSF (IL4-DC) some studies suggest the use of IFN-DC (IFN-α + GM-CSF). These cells exhibit a combined phenotype of myeloid DC, plasmacytoid DC (pDC) and NK. Considering the mixed profile of IFN-DCs alternative protocols can bring novel elements for immunotherapy. Monocytes isolated from HIV-infected patients were cultured in the presence of GM-CSF and IL-4 or IFN-α. On day 5 DCs were pulsed with AT-2-inactivated HIV and stimulated for 48 hours with a cocktail of proinflammatory cytokines. We assessed IFN-DC surface markers expression and T cell activation by flow cytometry; IL-10 and IL-12 production by ELISA. IFN-DC showed activated morphological and phenotypic features during basal state of maturity and exhibited features of pDC and NK different from IL4-DC. The IFN-DC like IL4-DC were able to produce IL-12 and stimulated T cells. So, the IFN-DC were able to stimulate the T cells as well as IL4-DCs

Phenotypic and functional analysis of alpha-type1 polarized dendritic cells (alpha-DC1) derived from monocytes of HIV-1 infected subjects

Resumo: A imunoterapia baseada em células dendríticas derivadas de monócitos (MoDCs) constitui uma estratégia promissora para o manejo de indivíduos infectados pelo HIV podendo atuar como um tratamento complementar para pacientes que fazem uso de terapia antirretroviral. Protocolos para obtenção de MoDCs convencionais, aqui denominadas IL4-DCs, utilizam como mediadores da diferenciação celular as citocinas IL-4 e GM-CSF que originam DCs imaturas com características mielóides. O estímulo das DCs imaturas com citocinas pró-inflamatórias TNF-alfa, IL-1beta e IL-6 ativa as MoDCs tornando-as aptas a apresentar antígenos e iniciar uma resposta imune específica. As IL4-DCs tem demonstrado capacidade de induzir resposta específica de linfócitos T, mas ainda demonstram baixo potencial de migrar para os linfonodos e produzir IL-12p70, essencial para estimular linfócitos Th1. Um protocolo alternativo tem proposto a utilização de interferons dos tipos I e II, além de agonistas de TLR para suplementar o estímulo para ativação de MoDCs. As MoDCs assim estimuladas são denominadas células dendríticas polarizantes para o tipo alfa-1 (alfa-DC1) e apresentam capacidade de produzir níveis elevados de IL-12p70 levando a uma potente resposta Th1. O potencial terapêutico de alfa-DC1 foi demonstrado inicialmente em protocolos de imunoterapia para pacientes de tumores e mais recentemente foi desenvolvido protocolo para indivíduos infectados pelo HIV. Há alguns anos nosso grupo de pesquisa está envolvido com protocolo clínico de imunoterapia baseada em IL4-DCs pulsadas com vírus quimicamente inativado. Neste contexto, com o objetivo de buscar alternativas para aperfeiçoar a imunoterapia, a nossa proposta foi avaliar in vitro aspectos fenotípicos e funcionais das alfa-DC1 pulsadas com HIV quimicamente inativado com potencial de aplicação na imunoterapia. Para tanto, foram avaliadas alfa-DC1 e IL4-DCs de pacientes e controle quanto à morfologia, expressão de moléculas de superfície, produção de IL-12p70, capacidade de estimular a produção de IFN-y e expressão de CD107a em linfócitos T autólogos e capacidade de estimular a proliferação de linfócitos T alogênicos. Os resultados revelaram que as alfa-DC1 apresentam maior expressão de CD40, CCR7 e CCR5, quando comparadas às IL4-DCs. Os resultados dos ensaios funcionais demonstraram que as alfa-DC1 tendem a produzir maiores níveis de IL-12p70 e que ambas alfa-DC1 e IL4-DCs foram capazes de estimular linfócitos T autólogos de pacientes a produzirem IFN-y. Ainda, ambas MoDCs foram capazes de induzir a proliferação de linfócitos T alogênicos de pacientes e HIV-. Embora as alfa-DC1 e IL4-DCs constituam populações distintas de MoDCs, demonstrando potencial de produção de IL-12p70 distintos, observamos que a capacidade de reconhecer e apresentar antígenos virais e estimular resposta de linfócitos T foi semelhante entre estas duas populaçõesAbstract: Immunotherapy based on monocyte-derived dendritic cells (MoDCs) has been shown to be a promising tool for treating HIV-infected individuals, and can act as a complementary treatment for HIV-infected individuals in use of antiretroviral therapy. Protocols for obtaining conventional MoDCs, here called IL4-DCs, use as mediators of cell differentiation the IL-4 and GM-CSF cytokines that originate immature DCs with myeloid characteristics. Stimulation with TNF-alpha, IL-1beta and IL-6 pro-inflammatory cytokines activates MoDCs making them able to present antigens and initiate a specific immune response. IL4-DCs have shown able to induce specific-T cell response, but still unable to migrate to lymph nodes and increase IL-12p70 production, essential to stimulate Th1 cells. An alternative protocol has proposed the use of type I and II interferons, in addition to TLR agonists to supplement the activation stimulus of MoDCs. These MoDCs are called alpha-type-1 polarized DCs (alpha-DC1), which have an exuberant capacity to produce high levels of IL-12p70 cytokine and to induce a potent Th1 response, consequently. The therapeutic potential of alpha-DC1 was first demonstrated in immunotherapy protocols for tumor patients and more recently a protocol has been developed for HIV-infected individuals. For some years, our research group has been involved in a clinical immunotherapy protocol based on IL4-DCs stimulated with chemically-inactivated virus. In this context, the aim of this study was to investigate alternatives to improve immunotherapy with proposing to evaluate in vitro phenotypic and functional aspects of alpha-DC1 pulsed with chemically inactivated HIV prospecting as a potential application in HIV immunotherapy. For that, alphaDC1 and IL4-DCs of HIV+ and HIV- subjects were evaluated for morphology, surface molecule expression, IL-12p70 production, ability to stimulate IFN-y production and CD107a expression in autologous T lymphocytes and ability to stimulate the proliferation of allogeneic T lymphocytes. The results revealed that alpha-DC1. The results of functional assays demonstrate that alpha-DC1 tends to produce higher levels of IL-12p70 and that both alpha-DC1 and IL4-DCs were able to stimulate IFN-y producing autologous T lymphocytes of HIV+ subjects. Also, both of MoDCs were able to induce allogeneic T lymphocytes proliferation of HIV+ and HIV- subjects. Although alpha-DC1 and IL4-DCs are distinct MoDCs populations that demonstrated different potential to release IL-12p70, we observed that the ability to recognize and present viral antigens and stimulate T lymphocyte response was similar between these two population

Phenotypic and functional profile of IFN-α-differentiated dendritic cells (IFN-DCs) from HIV-infected individuals

Dendritic cell (DC)-based immunotherapy is a promising strategy for the treatment of HIV-infected individuals. Different from the conventional protocol for DC differentiation based on the cytokine IL-4 (IL4-DCs), several studies have suggested obtaining DCs by culturing monocytes with type I IFN (IFN-α) to yield IFN-DCs, as performed in cancer therapy. To evaluate the phenotypic and functional characteristics, monocytes from HIV-infected subjects were differentiated into IFN-DCs or IL4-DCs, pulsed with chemically inactivated HIV and stimulated with pro-inflammatory cytokines. A comparative analysis between both types of monocyte-derived DCs (MoDCs) showed that immature IFN-DCs were phenotypically distinct from immature IL4-DCs at the baseline of differentiation, presenting a pre-activated profile. From the functional profile, we determined that IFN-DCs were capable of producing the cytokine IL-12 p70 and of inducing the production of IFN-γ by CD4 + T lymphocytes but not by TCD8+ lymphocytes. Our results suggest that IFN-DCs derived from HIV-infected individuals are able to recognize and present viral antigens to induce TCD4+ cellular immunity to HIV